Jennifer M. Campbell

De Novo Sequencing of Peptides Using MALDI/TOF-TOF

The recently developed MALDI TOF-TOF instrument yields relatively complex but interpretable fragmentation spectra. When coupled with a straightforward sequence extension algorithm, it is possible to develop complete peptide sequences de novo from the spectra. This approach has been applied to a set of peptides derived from typtic digestion of electrophoretically separated sea urchin egg membrane proteins. When directed to proteins that have been described previously, the results were in essential agreement with those obtained by conventional data base searching approaches, with certain important exceptions. The present method detected errors in published sequences and was able to develop sequences from peptides differing in mass by one dalton (Da). These results show both the power of the present approach and the need for using de novo methods more frequently than may be otherwise appreciated.

Matrix-assisted laser desorption/ionization-tandem mass spectrometry with high resolution and sensitivity for identification and characterization of proteins

Although peptide mass fingerprinting is currently the method of choice to identify proteins, the number of proteins available in databases is increasing constantly, and hence, the advantage of having sequence data on a selected peptide, in order to increase the effectiveness of database searching, is more crucial. Until recently, the ability to identify proteins based on the peptide sequence was essentially limited to the use of electrospray ionization tandem mass spectrometry (MS) methods. The recent development of new instruments with matrix‐assisted laser desorption/ionization (MALDI) sources and true tandem mass spectrometry (MS/MS) capabilities creates the capacity to obtain high quality tandem mass spectra of peptides. In this work, using the new high resolution tandem time of flight MALDI‐(TOF/TOF) mass spectrometer from Applied Biosystems, examples of successful identification and characterization of bovine heart proteins (SWISS‐PROT entries: P02192, Q9XSC6, P13620) separated by two‐dimensional electrophoresis and blotted onto polyvinylidene difluoride membrane are described. Tryptic protein digests were analyzed by MALDI‐TOF to identify peptide masses afterward used for MS/MS. Subsequent high energy MALDI‐TOF/TOF collision‐induced dissociation spectra were recorded on selected ions. All data, both MS and MS/MS, were recorded on the same instrument. Tandem mass spectra were submitted to database searching using MS‐Tag or were manually de novo sequenced. An interesting modification of a tryptophan residue, a “double oxidation”, came to light during these analyses.

Tandem time-of-flight mass spectrometry.

A new tandem time-of-flight (TOF-TOF) instrument has been developed by modifying a standard matrix-assisted laser desorption ionization (MALDI)-TOF instrument to make high-performance, high-energy collision-induced dissociation (CID) MALDI tandem mass spectrometry (MS) a practical reality. To optimize fragment spectra quality, the selected precursor ion is decelerated before entering a floating collision cell and the potential difference between the source and the collision cell defines the collision energy of the ions. Standard operating conditions for tandem MS use a 1-kV collision energy with single-collision conditions and increased laser power for ion formation. Hence, both high- and low-energy fragments are observed in MALDI TOF-TOF spectra. On standard peptides, sensitivities down to 1 fmol are demonstrated. On a mixture of two solution tryptic digests at the 25-fmol level, 23 spectra were sufficient to result in proper database identification.

The Liver Toxicity Biomarker Study: Phase I Design and Preliminary Results

Drug-induced liver injury (DILI) is the primary adverse event that results in withdrawal of drugs from the market and a frequent reason for the failure of drug candidates in development. The Liver Toxicity Biomarker Study (LTBS) is an innovative approach to investigate DILI because it compares molecular events produced in vivo by compound pairs that (a) are similar in structure and mechanism of action, (b) are associated with few or no signs of liver toxicity in preclinical studies, and (c) show marked differences in hepatotoxic potential. The LTBS is a collaborative preclinical research effort in molecular systems toxicology between the National Center for Toxicological Research and BG Medicine, Inc., and is supported by seven pharmaceutical companies and three technology providers. In phase I of the LTBS, entacapone and tolcapone were studied in rats to provide results and information that will form the foundation for the design and implementation of phase II. Molecular analysis of the rat liver and plasma samples combined with statistical analyses of the resulting datasets yielded marker analytes, illustrating the value of the broad-spectrum, molecular systems analysis approach to studying pharmacological or toxicological effects.

The identification of protein-protein interactions of the nuclear pore complex of Saccharomyces cerevisiae using high throughput matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry.

Mass spectrometry has become the technology of choice for detailed identification of proteins in complex mixtures. Although electrophoretic separation, proteolytic digestion, mass spectrometric analysis of unseparated digests, and database searching have become standard methods in widespread use, peptide sequence information obtained by collision-induced dissociation and tandem mass spectrometry is required to establish the most comprehensive and reliable results. Most tandem mass spectrometers in current use employ electrospray ionization. In this work a novel tandem mass spectrometer employing matrix-assisted laser desorption ionization-time-of-flight/time-of-flight operating at 200 Hz has been used to identify proteins interacting with known nucleoporins in the nuclear pore complex of Saccharomyces cerevisiae. Proteins interacting with recombinant proteins as bait were purified from yeast extracts and then separated by one-dimensional SDS-PAGE. Although peptide mass fingerprinting is sometimes sufficient to identify proteins, this study shows the importance of employing tandem mass spectrometry for identifying proteins in mixtures or as covalently modified forms. The rules for incorporating these features into MS-Tag are presented. In addition to providing an evaluation of the sensitivity and overall quality of collision-induced dissociation spectra obtained, standard conditions for ionization and fragmentation have been selected that would allow automatic data collection and analysis, without the need to adjust parameters in a sample-specific fashion. Other considerations essential for successful high throughput protein analysis are discussed.

Semi-targeted plasma proteomics discovery workflow utilizing two-stage protein depletion and off-line LC-MALDI MS/MS.

A quantitative proteomics workflow was implemented that provides extended plasma protein coverage by extensive protein depletion in combination with the sensitivity and breadth of analysis of two-dimensional LC-MS/MS shotgun analysis. Abundant proteins were depleted by a two-stage process using IgY and Supermix depletion columns in series. Samples are then extensively fractionated by two-dimensional chromatography with fractions directly deposited onto MALDI plates. Decoupling sample fractionation from mass spectrometry facilitates a targeted MS/MS precursor selection strategy that maximizes measurement of a consistent set of peptides across experiments. Multiplexed stable isotope labeling provides quantification relative to a common reference sample and ensures an identical set of peptides measured in the set of samples (set of eight) combined in a single experiment. The more extensive protein depletion provided by the addition of the Supermix column did not compromise overall reproducibility of the measurements or the ability to reliably detect changes in protein levels between samples. The implementation of this workflow is presented for a case study aimed at generating molecular signatures for prediction of first heart attack.

The Liver Toxicity Biomarker Study Phase I: Markers for the Effects of Tolcapone or Entacapone

The Liver Toxicity Biomarker Study is a systems toxicology approach to discover biomarkers that are indicative of a drug’s potential to cause human idiosyncratic drug-induced liver injury. In phase I, the molecular effects in rat liver and blood plasma induced by tolcapone (a “toxic” drug) were compared with the molecular effects in the same tissues by dosing with entacapone (a “clean” drug, similar to tolcapone in chemical structure and primary pharmacological mechanism). Two durations of drug exposure, 3 and 28 days, were employed. Comprehensive molecular analysis of rat liver and plasma samples yielded marker analytes for various drug–vehicle or drug–drug comparisons. An important finding was that the marker analytes associated with tolcapone only partially overlapped with marker analytes associated with entacapone, despite the fact that both drugs have similar chemical structures and the same primary pharmacological mechanism of action. This result indicates that the molecular analyses employed in the study are detecting substantial “off-target” markers for the two drugs. An additional interesting finding was the modest overlap of the marker data sets for 3-day exposure and 28-day exposure, indicating that the molecular changes in liver and plasma caused by short- and long-term drug treatments do not share common characteristics.

CHAPTER 11:Discovery and Validation Case Studies, Recommendations: Discovery and Development of Multimarker Panels for Improved Prediction of Near‐Term Myocardial Infarction

This report is a case study of the complex process of designing and performing a biomarker discovery study, validating its preliminary findings, and translating these into a clinical utility that would be suitable for the requirements of FDA clearance. The process is illustrated on the development of a multianalyte (protein) panel that can significantly enhance the prediction of the risk of near‐term myocardial infarction (MI) over traditional risk factors. The utilized clinical material is a subset of the Copenhagen General Population Study consisting of a matched set 252 cases, defined as fatal or nonfatal MI within four years of blood collection, and 499 controls. For discovery, we utilized high‐resolution mass‐spectrometric profiling of pooled samples complemented with multiplexed immunoassays. Discoveries were validated by using multiple reaction monitoring (MRM) liquid chromatography‐mass spectrometry (LC‐MS). It was demonstrated that a six‐protein panel comprised of analytes measured by MRM and immunoassays could significantly improve risk prediction over that provided by traditional risk factors alone. The path of developing a “submission package” for FDA clearance is also discussed.